Disruption of entire Cables2 locus leads to embryonic lethality by diminished Rps21 gene expression and enhanced p53 pathway.

In vivo function of CDK5 and Abl enzyme substrate 2 (Cables2), belonging to the Cables protein family, is unknown. Here, we found that targeted disruption of the entire Cables2 locus (Cables2d) caused growth retardation and enhanced apoptosis at the gastrulation stage and then induced embryonic lethality in mice. Comparative transcriptome analysis revealed disruption of Cables2, 50% down-regulation of Rps21 abutting on the Cables2 locus, and up-regulation of p53-target genes in Cables2d gastrulas. We further revealed the lethality phenotype in Rps21-deleted mice and unexpectedly, the exon 1-deleted Cables2 mice survived. Interestingly, chimeric mice derived from Cables2d ESCs carrying exogenous Cables2 and tetraploid wild-type embryo overcame gastrulation. These results suggest that the diminished expression of Rps21 and the completed lack of Cables2 expression are intricately involved in the embryonic lethality via the p53 pathway. This study sheds light on the importance of Cables2 locus in mouse embryonic development.

Generation of CRISPR/Cas9-mediated bicistronic knock-in ins1-cre driver mice.

In the present study, we generated novel cre driver mice for gene manipulation in pancreatic ß cells. Using the CRISPR/Cas9 system, stop codon sequences of Ins1 were targeted for insertion of cre, including 2A sequences. A founder of C57BL/6J-Ins1(em1 (cre) Utr) strain was produced from an oocyte injected with pX330 containing the sequences encoding gRNA and Cas9 and a DNA donor plasmid carrying 2A-cre. (R26GRR x C57BL/6J-Ins1(em1 (cre) Utr)) F1 mice were histologically characterized for cre-loxP recombination in the embryonic and adult stages; cre-loxP recombination was observed in all pancreatic islets examined in which almost all insulin-positive cells showed tdsRed fluorescence, suggesting ß cell-specific recombination. Furthermore, there were no significant differences in results of glucose tolerance test among genotypes (homo/hetero/wild). Taken together, these observations indicated that C57BL/6J-Ins1(em1 (cre) Utr) is useful for studies of glucose metabolism and the strategy of bicistronic cre knock-in using the CRISPR/Cas9 system could be useful for production of cre driver mice.

Germline recombination in a novel Cre transgenic line, Prl3b1-Cre mouse.

Spermatogenesis is a complex and highly regulated process by which spermatogonial stem cells differentiate into spermatozoa. To better understand the molecular mechanisms of the process, the Cre/loxP system has been widely utilized for conditional gene knockout in mice. In this study, we generated a transgenic mouse line that expresses Cre recombinase under the control of the 2.5 kbp of the Prolactin family 3, subfamily b, member 1 (Prl3b1) gene promoter (Prl3b1-cre). Prl3b1 was initially reported to code for placental lactogen 2 (PL-2) protein in placenta along with increased expression toward the end of pregnancy. PL-2 was found to be expressed in germ cells in the testis, especially in spermatocytes. To analyze the specificity and efficiency of Cre recombinase activity in Prl3b1-cre mice, the mice were mated with reporter R26GRR mice, which express GFP ubiquitously before and tdsRed exclusively after Cre recombination. The systemic examination of Prl3b1-cre;R26GRR mice revealed that tdsRed-positive cells were detected only in the testis and epididymis. Fluorescence imaging of Prl3b1-cre;R26GRR testes suggested that Cre-mediated recombination took place in the germ cells with approximately 74% efficiency determined by in vitro fertilization. In conclusion, our results suggest that the Prl3b1-cre mice line provides a unique resource to understand testicular germ-cell development. genesis 54:389-397, 2016. © 2016 Wiley Periodicals, Inc.

Combined Overexpression of JARID2, PRDM14, ESRRB, and SALL4A Dramatically Improves Efficiency and Kinetics of Reprogramming to Induced Pluripotent Stem Cells.

Identification of a gene set capable of driving rapid and proper reprogramming to induced pluripotent stem cells (iPSCs) is an important issue. Here we show that the efficiency and kinetics of iPSC reprogramming are dramatically improved by the combined expression of Jarid2 and genes encoding its associated proteins. We demonstrate that forced expression of JARID2 promotes iPSC reprogramming by suppressing the expression of Arf, a known reprogramming barrier, and that the N-terminal half of JARID2 is sufficient for such promotion. Moreover, JARID2 accelerated silencing of the retroviral Klf4 transgene and demethylation of the Nanog promoter, underpinning the potentiating activity of JARID2 in iPSC reprogramming. We further show that JARID2 physically interacts with ESRRB, SALL4A, and PRDM14, and that these JARID2-associated proteins synergistically and robustly facilitate iPSC reprogramming in a JARID2-dependent manner. Our findings provide an insight into the important roles of JARID2 during reprogramming and suggest that the JARID2-associated protein network contributes to overcoming reprogramming barriers.

Truncated Cables1 causes agenesis of the corpus callosum in mice.

Agenesis of the corpus callosum (ACC) is a congenital abnormality of the brain structure. More than 60 genes are known to be involved in corpus callosum development. However, the molecular mechanisms underlying ACC are not fully understood. Previously, we produced a novel transgenic mouse strain, TAS, carrying genes of the tetracycline-inducible expression system that are not involved in brain development, and inherited ACC was observed in the brains of all homozygous TAS mice. Although ACC was probably induced by transgene insertion mutation, the causative gene and the molecular mechanism of its pathogenesis remain unclear. Here, we first performed interphase three-color fluorescence in situ hybridization (FISH) analysis to determine the genomic insertion site. Transgenes were inserted into chromosome 18 ∼12.0 Mb from the centromere. Gene expression analysis and genomic PCR walking showed that the genomic region containing exon 4 of Cables1 was deleted by transgene insertion and the other exons of Cables1 were intact. The mutant allele was designated as Cables1(TAS). Interestingly, Cables1(TAS) mRNA consisted of exons 1-3 of Cables1 and part of the transgene that encoded a novel truncated Cables1 protein. Homozygous TAS mice exhibited mRNA expression of Cables1(TAS) in the fetal cerebrum, but not that of wild-type Cables1. To investigate whether a dominant negative effect of Cables1(TAS) or complete loss of function of Cables1 gives rise to ACC, we produced Cables1-null mutant mice. ACC was not observed in Cables1-null mutant mice, suggesting that a dominant negative effect of Cables1(TAS) impairs callosal formation. Moreover, ACC frequency in Cables1(+/TAS) mice was significantly lower than that in Cables1(-/TAS) mice, indicating that wild-type Cables1 interfered with the dominant negative effect of Cables1(TAS). This study indicated that truncated Cables1 causes ACC and wild-type Cables1 contributes to callosal formation.

Exome sequencing of senescence-accelerated mice (SAM) reveals deleterious mutations in degenerative disease-causing genes.

BACKGROUND: Senescence-accelerated mice (SAM) are a series of mouse strains originally derived from unexpected crosses between AKR/J and unknown mice, from which phenotypically distinct senescence-prone (SAMP) and -resistant (SAMR) inbred strains were subsequently established. Although SAMP strains have been widely used for aging research focusing on their short life spans and various age-related phenotypes, such as immune dysfunction, osteoporosis, and brain atrophy, the responsible gene mutations have not yet been fully elucidated. RESULTS: To identify mutations specific to SAMP strains, we performed whole exome sequencing of 6 SAMP and 3 SAMR strains. This analysis revealed 32,019 to 38,925 single-nucleotide variants in the coding region of each SAM strain. We detected Ogg1 p.R304W and Mbd4 p.D129N deleterious mutations in all 6 of the SAMP strains but not in the SAMR or AKR/J strains. Moreover, we extracted 31 SAMP-specific novel deleterious mutations. In all SAMP strains except SAMP8, we detected a p.R473W missense mutation in the Ldb3 gene, which has been associated with myofibrillar myopathy. In 3 SAMP strains (SAMP3, SAMP10, and SAMP11), we identified a p.R167C missense mutation in the Prx gene, in which mutations causing hereditary motor and sensory neuropathy (Dejerine-Sottas syndrome) have been identified. In SAMP6 we detected a p.S540fs frame-shift mutation in the Il4ra gene, a mutation potentially causative of ulcerative colitis and osteoporosis. CONCLUSIONS: Our data indicate that different combinations of mutations in disease-causing genes may be responsible for the various phenotypes of SAMP strains.

Sirt1, p53, and p38(MAPK) are crucial regulators of detrimental phenotypes of embryonic stem cells with Max expression ablation.

c-Myc participates in diverse cellular processes including cell cycle control, tumorigenic transformation, and reprogramming of somatic cells to induced pluripotent cells. c-Myc is also an important regulator of self-renewal and pluripotency of embryonic stem cells (ESCs). We recently demonstrated that loss of the Max gene, encoding the best characterized partner for all Myc family proteins, causes loss of the pluripotent state and extensive cell death in ESCs strictly in this order. However, the mechanisms and molecules that are responsible for these phenotypes remain largely obscure. Here, we show that Sirt1, p53, and p38(MAPK) are crucially involved in the detrimental phenotype of Max-null ESCs. Moreover, our analyses revealed that these proteins are involved at varying levels to one another in the hierarchy of the pathway leading to cell death in Max-null ESCs.

ALEX1 suppresses colony formation ability of human colorectal carcinoma cell lines.

Arm protein lost in epithelial cancers, on chromosome X (ALEX; also known as armadillo repeat containing, X-linked [ARMCX]) is a novel subgroup within the armadillo (ARM) family, which has several ARM repeat domains. The biological function of classical ARM family members such as ß-catenin is well understood, but that of the ALEX/ARMCX family members is largely unknown. Here we evaluate the effects of ALEX1 overexpression on in vitro colony formation ability and expression of ALEX1 mRNA in human colorectal tumor. Overexpression of ALEX1 suppressed the anchorage-dependent and -independent colony formation of human colorectal carcinoma cell lines by the study of stable clones of HCT116 cells expressing ALEX1 protein. Bisulfite genomic sequencing revealed that the promoter region of ALEX1 gene was highly methylated in both HCT116 and SW480 cells in comparison with PANC-1 and MCF-7 cells, which express endogenous ALEX1 mRNA, indicating the capability of promoter methylation to silence ALEX1 gene in HCT116 and SW480 cells. Our current findings suggest that overexpression of ALEX1 play a negative role in human colorectal tumorigenesis.

Reconstruction system of rat lymphatic tracts after total iliac lymphadenectomy.

BACKGROUND/AIMS: The lymphatic network is capable of reconstruction after the regional lymphadenectomy; however, the biological reconstruction system has not been fully elucidated. Our aim is to evaluate the reconstruction system after bilateral iliac lymphadenectomy using rat models and to establish and characterize rat lymphatic endothelial cells (rLECs) from the reconstructed tracts, compared with those from pre-existing vessels. METHODOLOGY: Under general anesthesia, we excised the dyed bilateral lymphatic vessels. We reopened the treated rats again after 2-10 weeks to observe the reconstruction system. We isolated and cultured rLECs from afferent, efferent and reconstructed lymphatic vessels. We characterized them morphologically and biologically, and examined their activity of proliferation, migration and invasion. RESULTS: We demonstrated that lymph vessels were reconstructed at 4-10 weeks at the site of surgical removal of iliac lymphatic channels, which occurred mainly at the left side of abdomen of the rat (86% on the left side). Isolated cells were all confirmed to be rLECs by expression of lymphatic vessel-specific markers. Proliferative activity of reconstructed rLECs was significantly higher than the other two strains of rLECs. CONCLUSIONS: We first established the reconstruction system after rat total iliac lymphadenectomy and characterized rLECs from the three strains of lymphatic vessels.

A novel locus on proximal chromosome 18 associated with agenesis of the corpus callosum in mice.

Agenesis of the corpus callosum (ACC) is a congenital abnormality of the brain structure. We have produced transgenic mice expressing both reverse tetracycline-controlled transactivator (rtTA) and transcriptional silencer (tTS) ubiquitously. Although the transgene products do not affect development of the mouse brain, one of the founder lines, TAS, showed ACC, suggesting transgenic disruption of endogenous gene(s). To identify the causative gene and its role in ACC, we performed pathological investigations of the brain and chromosomal mapping of foreign genes in TAS mice. Sixty-two percent of the heterozygous TAS mice showed ACC accompanied with formation of Probst bundles, as seen in human. Complete penetrance of ACC was observed in homozygous TAS mice. Furthermore, homozygous TAS fetuses revealed that ACC is a congenital anomaly. Moreover, axons of the corpus callosum were not repelled by the midline glial structures in TAS mice. These findings suggested that the causative gene for ACC is involved in critical steps in corpus callosum development. Multiple FISH analyses were performed to determine the site of transgene insertion. On 1-color FISH analyses, rtTA and tTS were detected on the A/B region of chromosome 18, suggesting cointegration of the transgenes. On 2-color FISH analyses, tTS signal was observed in a region from 9.3 to 16.9 Mb on chromosome 18. The TAS mice may serve as a useful model to identify a novel gene regulating corpus callosum development and to gain a new insight into molecular genetics of ACC.

Human Arm protein lost in epithelial cancers, on chromosome X 1 (ALEX1) gene is transcriptionally regulated by CREB and Wnt/beta-catenin signaling.

The aberrant activation of Wnt signaling is a key process in colorectal tumorigenesis. Canonical Wnt signaling controls transcription of target genes via beta-catenin and T-cell factor/lymphoid enhancer factor family transcription factor complex. Arm protein lost in epithelial cancers, on chromosome X 1 (ALEX1) is a novel member of the Armadillo family which has two Armadillo repeats as opposed to more than six repeats in the classical Armadillo family members. Here we examine cis-regulatory elements and trans-acting factors involved in the transcriptional regulation of the ALEX1 gene. Site-directed mutations of a cyclic AMP response element (CRE) and an E-box impaired the basal activity of human ALEX1 promoter in colorectal and pancreatic cancer cell lines. Moreover, overexpression of CRE-binding protein (CREB) increased the ALEX1 promoter activity in these cell lines, whereas knockdown of CREB expression decreased the expression level of ALEX1 mRNA. Interestingly, luciferase reporter analysis and quantitative real-time RT-PCR demonstrated that the ALEX1 promoter was up-regulated in a CRE-dependent manner by continuous activation of Wnt/beta-catenin signaling induced by a glycogen synthase kinase-3 inhibitor and overexpression of beta-catenin. These results indicate that the CRE and E-box sites are essential cis-regulatory elements for ALEX1 promoter activity, and ALEX1 expression is regulated by CREB and Wntk/beta-catenin signaling.

Establishment and characterization of the human SaTM-1 anal canal squamous cell carcinoma cell line derived from lymph node metastasis.

Human anal canal squamous cell carcinoma (SCC) cell line has not yet been reported due to the rarity of this disease. Since cell lines to study this malignancy were not available, we attempted to establish and characterize anal canal SCC cell line from primary culture of lymph node metastasis. Six sublines were cloned and isolated from parental cells. They were designated as SaTM-1A, B, C, D, E and F. The features of the six sublines were characterized by reverse transcription-PCR, chemosensitivity test to 5-Fu and CDDP, immunohistochemistry, cDNA microarray analysis and tumorigenicity using immunodeficient mice. All sublines were proliferated in multiple layers at an average doubling time of 24.5 h. VEGF-A, -B, VEGFR-1, -R3 and EGFR were expressed in all sublines, whereas VEGF-D and EGF were not detected in all. SaTM-1 was proven to retain the characteristics of SCC by detection of p63 and cytokeratin 5/6. The cytotoxic effects of 5-Fu were almost similar, although those of CDDP showed different behavior, which was divided into two groups (SaTM-1A, B, E and SaTM-1C, D, F). The differences in gene expression between two groups were analyzed according to susceptibility to cytotoxic effects of CDDP. Thirty-six genes were successfully identified, which may be potentially associated with CDDP resistance. SaTM-1 cells formed tumors easily in vivo, therefore all subclones had tumorigenic property. This is the first report of successful establishment and characterization of a human anal canal SCC cell line, which may provide beneficial resources for investigating the biological features of human anal canal SCC.

von Willebrand factor type D domain mutant of SVS-1/SUSD2, vWD(m), induces apoptosis in HeLa cells.

SVS-1/SUSD2 is a novel gene, which inhibits growth and reverses tumorigenic phenotypes of cancer cells in vitro. Here we report identification of a mutant of SVS-1, designated SVS-1-vWD(m), in which conserved amino acids GLLG at positions 591-594 in von Willebrand factor type D (vWD) domain are replaced by AAAA. As observed by laser confocal microscope, intracellular localization of the mutant protein has changed such that both the N-terminus and the C-terminus of SVS-1-vWD(m) were localized in the inner surface of the plasma membrane, whereas the N-terminus of SVS-1 was localized in the outer surface of the plasma membrane. Additionally, SVS-1-vWD(m) was processed much less efficiently and in a slightly different manner. In in vitro studies, adenovirus-mediated transduction of the SVS-1-vWD(m)gene induced growth suppression of HeLa cells in a dose-dependent manner, as the wild-type gene and inhibition of anchorage-independent growth. Of great interest is the finding that the mutant protein, vWD(m), but not the wild-type one induced apoptosis, as observed by nuclear as well as DNA fragmentation. Activation of caspase-3 and -9, but not caspase-8 or -12, was also demonstrated in vWD(m)-expressing cells. An inhibition of Akt phosphorylation, a major survival signaling component, also occurred in vWD(m)-expressing HeLa cells. Together these data suggest that vWD(m) induces apoptosis by inactivation of survival signaling component Akt and activation of caspase cascade (mitochondrial pathway) in HeLa cells. We propose SVS-1-vWD(m)as an alternative gene for use in developing new therapeutic strategies for the treatment of cancer.

Isolation of a novel mouse gene, mSVS-1/SUSD2, reversing tumorigenic phenotypes of cancer cells in vitro.

We report isolation of a novel tumor-reversing gene, tentatively named SVS-1, encoding a protein of 820 amino acids with localization on the plasma membrane as a type I transmembrane protein. The gene was found among those downregulated in the activated oncogene-v-K-ras-transformed NIH3T3 cells, Ki3T3, with tumorigenic phenotype. SVS-1 protein harbors several functional domains inherent to adhesion molecules. Histochemical staining of mouse tissues using antibody raised against the protein showed the expression of the protein in restricted regions and cells, for example, strongly positive in apical membranes of epithelial cells in renal tubules and bronchial tubes. The protein inducibly expressed in human fibrosarcoma HT1080 cells and cervical carcinoma HeLa cells was found to be localized primarily on the plasma membrane, as stained with antibodies against FLAG tag in the N-terminus and against the C-terminal peptide of the protein. Expression of the protein in cells induced a variety of biological effects on cancer cells: detachment from the substratum and aggregation of cells and growth inhibition in HeLa cells, but no inhibition in non-tumorigenic mouse NIH3T3 cells. Inhibition of clonogenicity, anchorage-independent growth, migration and invasion through Matrigel was also observed. Taken together these results suggest that the SVS-1 gene is a possible tumor-reversing gene.

Clinical significance of large tenascin-C spliced variant as a potential biomarker for colorectal cancer.

BACKGROUND: Tenascin-C is an extracellular matrix protein forming various types of spliced variants. Low molecule variants are transiently present, but large spliced variants are predominantly overexpressed in proliferative processes or tumorigenesis in some varieties of cancer. However, the detection of the plasma level of large tenascin-C spliced variant (L-Tn-CSV) in colorectal cancer (CRC) has not been clarified. This study was performed to validate elevated plasma L-Tn-CSV levels as a possible biomarker for CRC. MATERIALS AND METHODS: Plasma samples were obtained before resection and from time to time postoperatively and stored at -80 degrees C until assay. Plasma L-Tn-CSV levels were evaluated in patients with primary (n = 162) and with recurrent (n = 20) CRC, including 48 healthy volunteers, measured by ELISA. RESULTS: The average plasma L-Tn-CSV concentrations of patients with primary CRC were 5,260 +/- 3,243.3 pg/ml and of patients with recurrent CRC 4,106 +/- 2,261.1 pg/ml, which were significantly elevated in comparison with those of healthy volunteers (2,364.3 +/- 7,49.6). The sensitivity for detecting CRC using plasma L-Tn-CSV was 56.6%, based on the mean +/- 2 SD of the concentrations of healthy controls (3,863.5), which was significantly higher than CEA (40.1%) and CA19-9 (23.6%). No obvious associations were evident between plasma L-Tn-CSV status and values of CEA and CA19-9 respectively. Statistically significant differences in plasma L-Tn-CSV were observed depending on tumor depth, lymph node metastasis, and TNM stage. Negative conversions of plasma L-Tn-CSV levels 6 months after resection were significantly higher in the completely curative resection group than in the non-curative groups (P < 0.001). CONCLUSION: The plasma L-Tn-CSV may serve very well as a useful biomarker for tumor staging and postoperative monitoring of preoperatively positive CRC that is independent and exceeds conventional tumor markers.

Development of ELISA using recombinant antigens for specific detection of mouse parvovirus infection.

Nucleotide sequences of mouse parvovirus (MPV) isolate, named MPV/UT, and mouse minute virus (MMV) were analyzed and used for expressing recombinant proteins in E. coli. ELISA tests using recombinant major capsid protein (rVP2) and recombinant major non-structural protein (rNS1) as antigens were developed and their performance in serologic detection of rodent parvovirus infection was assessed. MPV-rVP2 and MMV-rVP2 ELISAs reacted specifically with anti-MPV and anti-MMV mouse sera, respectively. MMV-rNS1 antigen had a wide reaction range with antisera to rodent parvoviruses including MPV, MMV, Kilham rat virus (KRV) and H-1 virus. All mice oronasally infected with MPV were seropositive at 4 weeks post-infection in screening by ELISAs using MPV-rVP2 and MMV-rNS1 antigens, but were negative by conventional ELISA using whole MMV antigen. A contact transmission experiment revealed that transmission of MPV occurred up to 4 weeks post-infection, and all cage mates were seropositive in screening with MPV-rVP2 and MMV-rNS1 ELISAs. These results indicate that MPV-rVP2 and MMV-rVP2 are specific ELISA antigens which distinguish between MPV and MVM infection, while MMV-rNS1 antigen can be used in generic ELISA for a variety of rodent parvoviruses. The higher sensitivity of MPV-rVP2 ELISA than conventional ELISA for detecting seroconversion to MPV in oronasally infected mice as well as in cage mates suggests the usefulness of MPV-rVP2 ELISA in quarantine and microbiological monitoring of MPV infection in laboratory mice.

A novel subpopulation lacking Oct4 expression in the testicular side population.

We characterized murine spermatogonial stem cells (SSCs) using a multi-parameter selection strategy, combining Oct4 expression determined by monitoring green fluorescent protein (GFP) expression, and the testicular side population (SP) showing weak fluorescence on Hoechst 33342 dye staining, as markers of stem cell purification. Testicular cells were collected from Oct4/GFP transgenic mice and analyzed using a fluorescence-activated cell sorter (FACS). SP was detected in testicular cell suspensions at an average rate of 0.10%. Multicolor analysis indicated that 96% of SP cells were negative for Oct4. The cells did not express SSC marker genes, but expressed Bcrp1. While the main population was 93% positive for pyronin Y staining, this was limited to 51% in SP. We found a novel subpopulation with reduced RNA content lacking Oct4 expression in testicular SP. These results suggest that the cells isolated by FACS represent a novel population of SSCs in the G0 quiescent state.